As one of the most common neurological diseases in this country, epilepsy affects approximately 2.5 million Americans. Drugs used to treat epilepsy often target neurotransmission, and their design and use would be advanced by a better understanding of the mechanisms of neurotransmission. It is commonly assumed that neurotransmitters are only released by synaptic vesicle fusion. However, our recent studies suggest that GABA transporters reverse during seizures, resulting in GABA efflux. This "nonvesicular" GABA release inhibits neurons, and is also the target of a new class of anticonvulsants, including gabapentin and vigabatrin. The current proposal extends this work by using rat neurons to address the following unanswered questions. What is the source of nonvesicular GABA release: neurons or glia? This will be examined using neuronal vs. glial specific GABA transporter antagonists in pure neuron, pure glia or mixed cultures. How do changes in cytosolic [GABA] affect vesicular GABA release? The effect on vesicular and nonvesicular GABA release of increasing or decreasing cytosolic [GABA] will be compared. How commonly is nonvesicular GABA release modulated by anticonvulsants? The effects on nonvesicular GABA release of pregabalin, topiramate, levetiracetam, and tiagabine will be studied. How ubiquitous is nonvesicular GABA release? The role of nonvesicular GABA release will be studied in brain slices from the hippocampus, neocortex, striatum, cerebellum and medulla. Does the glutamate transporter reverse as easily as the GABA transporter? The threshold for nonvesicular glutamate release will be determined. We propose that the GABA transporter reverses as part of a fail-safe negative feedback system. If the glutamate transporter reversed so easily, it would lead to runaway excitotoxicity. The proposed experiments are designed to answer fundamental questions about the newly recognized role of the GABA transporter in inhibition of seizures, and the mechanism of action of anticonvulsants. A complete description of GABA transporter function should help define the pathophysiology of epilepsy, and lead to a better understanding of how and when the new anticonvulsants should be used.